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Identification of novel ssDNA and RNA coliphage in wastewater
(Montana State University - Bozeman, College of Agriculture, 2024) Little, Agusta Rio; Chairperson, Graduate Committee: Blake Wiedenheft
Bacteriophages (phages) are the most abundant biological entities on Earth. However, our understanding of their diversity is limited, with a vast gap in knowledge regarding single- stranded DNA (ssDNA) and RNA phages. This study addresses this gap by isolating and characterizing ssDNA and RNA coliphages from wastewater, a suspected rich source of these understudied phages. Traditional phage isolation methods favor double-stranded DNA (dsDNA) phages, resulting in the underrepresentation of ssDNA and RNA phages. To overcome this bias, we employed enrichment strategies using small molecules that inhibit dsDNA phage replication. Additionally, we utilized an RNase-A assay to identify potential RNA phage candidates. These enrichment techniques led to the isolation of a circular ssDNA phage (POI 1) and a ssRNA phage (POI 8). A combination of biochemical assays, sequencing, and microscopy techniques were utilized to characterize these phages. Overall, this work demonstrates the effectiveness of enrichment strategies for isolating ssDNA and RNA phages and underscores the importance of developing optimized techniques to unlock the true diversity of these understudied phage populations.
Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes
(Springer Science and Business Media LLC, 2023-10) Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Karunakaran, Simrithaa; Rau, Michael J.; Basore, Katherine; Pokhrel, Nilisha; Englander, Noah; Fitzpatrick, James A. J.; Bothner, Brian; Antony, Edwin
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.
Development and characterization of a novel isothermal DNA amplification reaction
(Montana State University - Bozeman, College of Engineering, 2021) Ozay, Burcu; Chairperson, Graduate Committee: Scott McCalla; This is a manuscript style paper that includes co-authored chapters.
Isothermal nucleic acid amplification chemistries are gaining popularity as nucleic acid detection tools that can replace the current gold standard methods, PCR and its derivatives, with their simplicity, speed and applicability to point-of-care applications. In this work, we have developed and characterized a novel isothermal amplification chemistry, ultrasensitive DNA amplification reaction (UDAR). UDAR differs from similar chemistries with its unique, biphasic response with a high-gain output that can be captured with a cell-phone camera. The switch-like, nonlinear characteristics provide a definitive on/off signal for potential use in applications such as molecular diagnostics and DNA circuits. Tunability of the reaction was explored and the relationship between thermodynamic properties of the reaction templates and the reaction output was established. Limitations on fluorescent staining of reaction components by two popular commercial nucleic acid stains, SYBR Green II and SYBR Gold, were determined for a more accurate evaluation of the reaction output and reaction product analysis. A mathematical model of the reaction output was built and outputs from three different UDAR templates were successfully simulated. This model revealed important information on reaction pathways and helped identify the impact of individual reaction events. A comprehensive literature review of enhancement strategies for isothermal amplification reactions was conducted to serve as a guide to improve and modify these reactions according to different needs and applications. Lastly, UDAR was applied to microRNA detection, which are putative biomarkers for diseases such as cancer, malaria, and traumatic brain injury. Five different miRNAs were successfully detected by UDAR, down to 10 fM concentration. UDAR-based miRNA quantification is possible, with linear calibration curves provided between 10fM and 1 nM. This work has significant contributions to the growing field of isothermal nucleic acid amplification based-molecular detection systems by introducing a unique isothermal amplification chemistry, establishing design and manipulation techniques, and guiding improvement efforts of these technologies.
Integrating DNA fingerprinting of invasive watermilfoil strains into aquatic vegetation monitoring and assessment
(Montana State University - Bozeman, College of Agriculture, 2021) Gannon, Kathryn Alta; Chairperson, Graduate Committee: Ryan Thum; Raymond Newman and Ryan Thum were co-authors of the article, 'Integrating DNA fingerprinting of invasive watermilfoil strains into aquatic vegetation monitoring and assessment' submitted to the journal 'Journal of aquatic plant management' which is contained within this thesis.
Eurasian watermilfoil (Myriophyllum spicatum L.) and its hybrids with native northern watermilfoil (Myriophyllum spicatum L. x Myriophyllum sibiricum) (invasive watermilfoils) are among the most heavily managed invasive aquatic plants in the United States. Within invasive watermilfoils there are distinct strains which are produced through sexual reproduction and can be maintained indefinitely by clonal propagation. Different strains can differ in their invasiveness (e.g., growth and potential for spread) and response to herbicides. However, strain identification is not routinely implemented as part of management planning or evaluation. In this thesis, we integrated molecular fingerprinting that can distinguish different Eurasian and hybrid watermilfoil strains into aquatic vegetation monitoring in eight Minnesota lakes over the course of three years. We provide proof of concept that by tracking strain incidence over time it is possible 1) to detect changes in strain composition of invasive watermilfoil populations, and 2) to identify priority strains for future growth and herbicide assays. This study demonstrates that integrating genetic fingerprinting into aquatic vegetation management and evaluation holds promise to inform management decisions by identifying those strains that have the most invasive potential. As far as we are aware, this is the first published study to examine the strain composition of invasive watermilfoil populations over time.
Use of eDNA to estimate abundances of spawning Yellowstone cutthroat trout in Yellowstone National Part, Wyoming, USA
(Montana State University - Bozeman, College of Letters & Science, 2020) Detjens, Colleen Rachel; Chairperson, Graduate Committee: Alexander V. Zale
Invasive Lake Trout Salvelinus namaycush and whirling disease have reduced the abundance of native Yellowstone Cutthroat Trout Oncorhynchus clarkii bouvieri (YCT) in Yellowstone Lake, Yellowstone National Park, thereby disrupting the Yellowstone Lake ecosystem. One indication of the YCT population decline is the decrease in the number of adults returning to tributaries to spawn each spring. Yellowstone National Park implemented a gillnetting program to remove Lake Trout starting in 1995 to restore YCT abundance and size structure and thereby conserve the Yellowstone Lake ecosystem. An important metric for evaluating the success of the program is the number of YCT ascending spawning tributaries each year. Annually, 9 to 11 of these tributaries are visually surveyed on a weekly basis from May through July for the presence of spawners, but these surveys are time consuming. The use of environmental DNA (eDNA) has become increasingly common for determining presence of aquatic species and may provide managers with a more efficient tool for estimating abundances of YCT spawners. The primary objective of my study was to evaluate the efficacy and accuracy of using eDNA to detect the presence and estimate abundance of YCT spawners by collecting eDNA samples from spawning tributaries to Yellowstone Lake in conjunction with visual surveys of YCT spawners. A secondary objective was to evaluate whether terrestrial or semi-terrestrial species such as grizzly bear Ursus arctos horribilis and North American river otter Lontra canadensis could be detected in a water sample from YCT spawning tributaries. Environmental DNA quantities were more effective for determining presence of YCT spawners than for predicting their abundances, but eDNA quantities were positively related to spawner abundances. The difference between eDNA rates when spawners are present versus absent may provide managers with an efficient method for monitoring YCT in tributaries throughout Yellowstone Lake basin. I also demonstrated that DNA from a terrestrial species, grizzly bear, can be detected in water samples. Incorporation of eDNA sampling with existing methods for monitoring YCT spawners in Yellowstone Lake tributaries would facilitate an increased scale of assessment and allow for detection and quantification of multiple species of current and future interest from single samples.
Fluorescence quenching in 2-aminopurine-labeled model DNA systems
(Montana State University - Bozeman, College of Letters & Science, 2019) Remington, Jacob Michael; Chairperson, Graduate Committee: Patrik R. Callis; Abbey M. Philip, Mahesh Hariharan and Bern Kohler were co-authors of the article, 'On the origin of multiexponential fluorescence decays from 2-aminopurine labeled dinucleotides' in the journal 'The journal of chemical physics' which is contained within this thesis.; Martin McCullagh and Bern Kohler were co-authors of the article, 'Molecular dynamics simulations of 2-aminopurine-labeled dinucleoside monophosphates reveal multiscale stacking kinetics' in the journal 'Journal of physical chemistry B' which is contained within this thesis.
For the last 50 years changes to the fluorescence properties of 2-aminopurine have been used to probe the structure and dynamics of DNA. 2-Aminopurine's utility has arisen from the quenching of its emission when pi-stacked with neighboring nucleobases. In the time-domain, the emission decay profile of 2-aminopurine requires multiple exponential decay components to model. Despite its extensive usage, the microscopic origin of the decay heterogeneity is not clear. In this thesis, steady-state absorption, fluorescence, and time-resolved fluorescence results are compared to multiple microsecond molecular dynamics simulations of 2-aminopurine-labeled adenine containing single-stranded DNA oligomers of varying length and position of the 2-aminopurine probe. First, previous reports of ultrafast electron transfer in pi-stacked adenine oligomers are used to build a new model for quenching of 2-aminopurine that is pi-stacked with adenine. For dinucleotides, a static distribution of unstacked structures combined with a distance dependent electron transfer mechanism is posited to explain the disperse emission decay timescales. Investigating the dinucleotides with molecular dynamics simulations analyzed with Markov state models quantify the structural heterogeneity of the dinucleotides. At least seven structures are sampled that could alter the quenching of 2-aminopurines's fluorescence. The Markov state models also demonstrate the timescales for transitions between these structures range from 1.6 to 25 ns, suggesting 2-aminopurine, with its monomer-like lifetime of 10 ns, is sensitive to the conformational dynamics of the dinucleotides as well. This dual fluorescence quenching and molecular dynamics simulation approach is extended to 2-aminopurine labeled trinucleotides and 15 base oligomers to interrogate the position dependent structural heterogeneity and conformational dynamics in these systems. Both shifts in the experimental absorption spectra, and molecular dynamics simulations agree that the interior base is more likely to be stacked than the exterior bases. Time-resolved emission experiments reveal emission from 2-aminopurine is quenched faster on the 5' end relative to the 3' end, in agreement with the faster stacking kinetics observed for bases on the 5' end relative to the 3' end obtained from molecular dynamics simulation. These results suggest that the time-resolved emission from 2-aminopurine may serve as an experimental observable for calibration of the dynamical properties predicted by molecular dynamics simulation.
Transduction of antigens into amplifiable DNA signals using structure switching aptamers
(Montana State University - Bozeman, College of Engineering, 2019) Kayalar, Canberk; Chairperson, Graduate Committee: Stephanie McCalla
Detection of specific antigens has one vital step in common: detection of biomarkers. Diagnostic testing that is rapid and reliable is unavailable in limited resource and rural settings. The solution to this need must be simple, inexpensive, robust, rapid and not require highly trained personnel to operate. Aptamers are capable of delivering those needs when matched with a novel high gain amplification method. This thesis focuses on important aspects of a novel protein detection assay that uses aptamers. Aspects that play an important role on the assay's success were investigated; aptamer selection and design of structure switching aptamers, designing DNA templates that will transduce the signal created by the aptamers, solid phase selection, aptamer immobilization on the solid phase, protein capture, and amplification of the signal. The first step was to find aptamers that were proven to specifically target clinically relevant targets and modify them to suit the needs of the assay. It is important to validate the aptamers' performance. The second important step was finding a solid phase that is compatible with the novel nucleotide amplification reaction that will be used to amplify the signal produced by the aptamers. Paramagnetic microbeads, membranes and polyacrylamide hydrogels were potential candidates for solid phases. Non-specific interaction of the target protein with the solid phase surface will not have negative effects while running the assay due to the structure switching of the aptamers however, it prevented the accurate quantification of the protein capture by aptamers. There is a need for the development of a blocking buffer that is specific to the solid phase. Washing of the excess DNA templates that are not bound to target-bound aptamers plays an important role in the assay's accuracy. The results presented here show the preliminary work that has been done for the novel protein detection assay that uses structure switching aptamers. This assay has the potential to detect diseases at point-of-care in low resource settings.
A mathematical model of a biphasic DNA amplification reaction
(Montana State University - Bozeman, College of Letters & Science, 2019) Ciesielski, Danielle Kristine; Chairperson, Graduate Committee: Tomas Gedeon
Isothermal DNA amplification reactions have many applications ranging from analyte detection to DNA circuits. EXPonential Amplification Reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This Ultrasensitive DNA Amplification Reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms. We show that the model can reproduce the experimentally observed biphasic behavior. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding caused by two palindromic trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. Understanding of these mechanisms also illuminates behavior of EXPAR and other nucleic acid amplification reactions. For a deeper understanding of the roles these mechanisms play in DNA amplification reactions, we apply dynamical systems analysis to the model. We first consider the long term behavior of partial models that lack key reaction mechanisms described above to see how their omission impacts the system's overall behavior. Then we use perturbation theory to examine the time scales on which these mechanisms operate and how their interaction leads to biphasic growth. We find that mechanisms (i) and (ii) together create a stable equilibrium reminiscent of EXPAR reactions, but the addition of mechanism (iii) changes the stability of this equilibrium and generates UDAR's characteristic high amplification. Finally, mechanism (iv) introduces a second stable equilibrium that indicates that polymerase sequestration is the mechanism that ends the second fast amplification phase. In addition, throughout this work we identify which rate constants shape different parts of the biphasic growth. These results can guide future work in rational design of molecular detection assays.
Excited-state dynamics of biological molecules in solution: photoinduced charge transfer in oxidatively damaged DNA and deactivation of violacein in viscous solvents
(Montana State University - Bozeman, College of Letters & Science, 2017) Beckstead, Ashley Ann; Chairperson, Graduate Committee: Robert Walker
UV radiation from the sun is strongly absorbed by DNA, and the resulting electronic excited states can lead to the formation of mutagenic photoproducts. Decades of research have brought to light the excited-state dynamics of single RNA and DNA nucleobases, but questions remain about the nature of excited states accessed in DNA strands. In this thesis, I present ultrafast spectroscopic observations of photoinduced electron transfer from the oxidatively damaged bases, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine, to adenine in three dinucleotides. The results reveal that charge transfer states are formed on a timescale faster than our instrumental resolution (<0.5 ps), and back electron transfer efficiently returns the excited-state population to the ground state on timescales from tens to hundreds of ps. In addition to recent spectroscopic observations of charge transfer state species in DNA by other groups, our results have augmented understanding of the long-lived transient signals observed in DNA strands. The observation of photoinduced electron transfer in these oxidatively damaged nucleobases also supports a recent proposal regarding the role of oxidative products in pre-RNA catalysis. I discuss these observations in the contexts of fundamental DNA excited-state dynamics and prebiotic chemical evolution. In this thesis, I also present the first ultrafast spectroscopic investigation of violacein, a pigment isolated from Antarctic bacteria. Despite claims for the photoprotective role of this pigment, there has never been a spectroscopic analysis of excited-state deactivation in violacein. Emission spectra, fluorescence quantum yields and excited-state lifetimes of violacein in various solvents were measured for the first time. Both the fluorescence quantum yield and excited-state lifetime of violacein increase in increasingly viscous solvents, suggesting a large-scale motion mediates excited-state deactivation. I compare these results to similar observations of viscosity-dependent excited-state decay rates in other molecules. I also consider the relevance of violacein's excited-state properties to the hypothesized sunscreening role of violacein. Overall, the studies presented in this dissertation illustrate how ultrafast spectroscopic techniques can be used to unravel complex biomolecular excited-state dynamics in solution.
Subnanosecond emission from model DNA oligomers characterized through time-correlated single-photon counting spectroscopy
(Montana State University - Bozeman, College of Letters & Science, 2017) Skowron, David John; Chairperson, Graduate Committee: Robert Walker; Yuyuan Zhang, Ashley A. Beckstead, Jacob M. Remington, Madison Strawn and Bern Kohler were co-authors of the article, 'Subnanosecond emission dynamics of AT DNA oligonucleotides' in the journal 'Journal of chemical physics and physical chemistry' which is contained within this thesis.
Exposure of DNA to UV radiation creates electronic excited states that can decay to mutagenic photoproducts. Excited states can return to the electron ground state through deactivation pathways, preventing photochemical damage. Understanding has significantly advanced over the last decade through the applications of time-resolved techniques capable of picosecond and femtosecond time-resolution. While significant strides have been made towards understanding monomeric deactivation pathways, unraveling the complex photophysics of base multimers still presents a significant challenge. This report uses time-resolved fluorescence and ultrafast transient absorbance to analyze model DNA oligomers to understand how fundamental interactions between monomeric constituents influences the dynamics of base multimers. Model single- and double-stranded DNA oligomers were investigated using the time correlated single photon counting technique to address the uncertainty over how to compare results from time-resolved fluorescent and transient absorption techniques. Emission lifetimes ranging from 50 to 200 ps quantitatively agree with lifetimes measured from transient absorption experiments indicating emission observed on timescales greater than a few picoseconds is the result of excimer or charge recombination luminescence. In attempts to further characterize the time-resolved emission from model oligomers adenine oligomers consisting of 2 and 18 base constituents were examined in aqueous water and heavy water solutions. Differences in dynamics between the two oligomers revealed the average number of bases present within a stacked domain influence the dynamics of these systems. Lifetimes of the emission decays were assigned excimer-like states with various degrees of charge-transfer character. Finally, to further demonstrate the importance of base stacking domain length on the dynamics of these systems, time-resolved emission and absorption of the adenine dinucleotide and 18-mer where examined at temperatures ranging from 7 °C - 80 °C. It was observed that the kinetics between the oligomers was noticeably different at lower temperatures, but not at higher temperatures. It was concluded the domain length of the 18-mer was similar to the domain length of the dinucleotide at high temperatures, but not at low temperatures, demonstrating the domain length significant impacts theS photophysics of DNA.

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